Project Summary/Abstract Modulation of inflammation in aging lung Increased severity and delayed recovery of pneumonia and associated lung injury in older patients represents a serious threat for this vulnerable population, but molecular mechanisms of this age-dependent phenomenon remain poorly understood. As antioxidant defense mechanisms in aging organism become impaired, increased production of reactive oxygen species during inflammation may cause exaggerated oxidant stress. In turn, increased oxidation of the cell membrane and circulating phospholipids leads to generation of a family of fragmented products of phospholipid oxidation (FPL), which may exhibit deleterious effects on the host cells. In pilot studies we used a mass spectrometry approach and identified several FPL species with highest levels found in the inflamed aging lungs. Our pilot studies suggest that age-dependent elevation of FPLs augments lung dysfunction and impairs vascular endothelial cell (EC) barrier in cell and animal models of acute lung injury (ALI). We hypothesize that increased generation of FPL in the aging population as a result of dysregulated redox balance exacerbates lung inflammation and vascular dysfunction via direct effects on endothelial permeability, FPL-induced activation of thioredoxin interacting protein, and activation of cell death mechanisms leading to impaired ALI recovery. Using clinically relevant models of ALI induced by live antibiotic- resistant Staphylococcus aureus (S. au, USA300 CA-MRSA clinical strain 923) or heat-killed S. au recapitulating clinical treatment of antibiotics-sensitive strains, this translational study will employ for the first time the quantitative mass spectrometry approach to identify specific FPL elevated in the aging lung during ALI and test their role in exacerbation of lung inflammation and barrier dysfunction. LC-MS-MS and imaging MS technologies will be applied to preclinical mouse models of inflammatory lung injury to identify the spectrum of FPL produced in the inflamed aging lungs, generate the maps of FPL distribution in the lung samples and relate them to topography of lung injury and ALI severity in young and aging lungs. Mechanistic will focus on thioredoxin interacting protein and cell death signaling to uncover molecular basis of FPL-exacerbated lung injury in the aging lungs. Finally, this study will use genetic and pharmacologic interventions in animal models to interrogate key pathologic mechanisms defining age-related exacerbation of lung inflammation. The results of this study will advance our understanding of pathologic mechanisms which determine more severe inflammation in the aging lungs and may open a new direction in controlling ALI/ARDS in the aging population by inactivating FPL-induced inflammatory cascades or preventing generation of deleterious FPL products. These findings can be used for development of protocols for advanced antioxidant and anti-inflammatory treatment of the aging population at risk.